Abstract
Ca currents were examined with regard to their recovery from inactivation. The experiments were done on isolated nerve cell bodies ofHelix aspersa using a combined suction pippet, microelectrode method for voltage clamp, and internal perfusion. Ca currents were separated by suppressing K and Na currents.
The time course of recovery was determined by applying a test pulse at intervals ranging from 1 msec to 20 sec after prepulses varying from 20 to 3000 msec in duration. Each pair of pulses was preceded by a control pulse to ensure that the Ca currents had recovered before the next test pair was applied. Ba and Ca currents were compared and the effects of intracellular perfusion with EGTA, ATP, and vanadate were examined.
Ba currents recovered in two stages and this time course was well fit by a sum of two exponentials with amplitudes and time constants given byA 1 andτ 1 for the fast component andA 2 andτ 2 for the slow component. In Ba the time constants were unchanged when prepulse durations were prolonged from 70 to 700 msec, although the initial amplitudesA 1 andA 2, particularlyA 2, were increased.
Comparable influxes of Ca during the prepulse caused much more inactivation, but interestingly the recovery occurred at the same rate. The time course of Ca current recovery was also fit by a sum of two exponentials, the time constants of which were both smaller than the time constants of Ba current recovery. However, the time constants of Ca current recovery were increased markedly when prepulse durations were prolonged. Increasing the extracellular Ca concentration had a similar effect.
Increasing the Ba influx had no effect on the recovery time constants, and the Ba results are consistent with reversible inactivation gating of potential-dependent membrane Ca channels. The Ca results show that Ca influx enhances inactivation. Intracellular perfusion with EGTA resulted in less inactivation in the cast of Ca but it had no effect on Ba currents. Intracellular ATP increased the rate of recovery of Ca currents, and intracellular vanadate inhibited recovery. It is concluded that recovery of Ca channels depends upon both Ca influx and membrane potential and is modulated by agents which affect Ca metabolism.
Key words: Ca current, voltage clamp, ATP, snail neuron
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